Genetically modified plants having modulated flower development

ABSTRACT

The present invention provides a genetically modified plant and a method for producing such a plant characterized as having modulated flower meristem development. As an illustrative example, the invention provides genetically modified tobacco and aspen plants characterized as having early floral meristem development and comprising a structural gene encoding the LEAFY protein in its genome.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/360,336 filed on Dec. 21, 1994., now U.S. Pat. No.5,637,785.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to plant genetic engineering, andspecifically to novel genetically engineered plants characterized ashaving a phenotype of early flower meristem development, and methods forproducing such plants.

2. Description of Related Art

Most angiosperm species are induced to flower in response toenvironmental stimuli such as day length and temperature, and internalcues, such as age. Adult organs of flowering plants develop from groupsof stem cells called meristems. The identity of a meristem is inferredfrom structures it produces: vegetative meristems give rise to roots andleaves, inflorescence meristems give rise to flower meristems, andflower meristems give rise to floral organs such as sepals and petals.Not only are meristems capable of generating new meristems of differentidentity, but their own identity can change during development. Forexample, a vegetative shoot meristem can be transformed into aninflorescence meristem upon floral induction, and in some species, theinflorescence meristem itself will eventually become a flower meristem.Despite the importance of meristem transitions in plant development,little is known about the underlying mechanisms.

Following germination, the shoot meristem produces a series of leafmeristems on its flanks. However, once floral induction has occurred,the shoot meristem switches to the production of flower meristems.Flower meristems produce floral organ primordia, which developindividually into sepals, petals, stamens or carpels. Thus, flowerformation can be thought of as a series of distinct developmental steps,i.e. floral induction, the formation of flower primordia and theproduction of flower organs. Mutations disrupting each of the steps havebeen isolated in a variety of species, suggesting that a genetichierarchy directs the flowering process (see for review, Weigel andMeyerowitz, In Molecular Basis of Morphogenesis (ed. M. Bemfield). 51stAnnual Symposium of the Society for Developmental Biology, pp. 93-107,New York, 1993).

Recently, studies of two distantly related dicotyledons, Arabidopsisthaliana and Antirrhinum majus, led to the identification of threeclasses of homeotic genes, acting alone or in combination to determinefloral organ identity (Bowman, et al., Development, 112: 1, 1991;Carpenter and Coen, Genes Devl., 4:1483, 1990; Schwarz-Sommer, et al.,Science, 250:931, 1990). Several of these genes are transcriptionfactors whose conserved DNA-binding domain has been designated the MADSbox (Schwarz-Sommer, et al., supra).

Earlier acting genes that control the identity of flower meristems havealso been characterized. Flower meristems are derived from inflorescencemeristems in both Arabidopsis and Antirrhinum. Two factors that controlthe development of meristematic cells into flowers are known. InArabidopsis, the factors are the products of the LEAFY gene (Weigel, etal., Cell 69:843, 1992) and the APETALA1 gene (Mandel, et al., Nature360:273, 1992, herein incorporated by reference in its entirety and SEQID NO:1 and 2). When either of these genes is inactivated by mutation,structures combining the properties of flowers and inflorescence develop(Weigel, et al., supra; Irish and Sussex, Plant Cell, 2:741, 1990). InAntirrhinum, the homologue of the Arabidopsis LEAFY gene is FLORICA ULA(Coen, et al., Cell, 63:1311, 1990) and that of the APETALA1 gene isSQUAMOSA (Huijser, et al., EMBO J, 11: 1239, 1992). The latter paircontains MADS box domains.

LEAFY is expressed very early in floral anlagen and floral primordia,consistent with it having a direct role in establishing floral meristemidentity. In the developing floral primordium, LEAFY expression isdetected much earlier than expression of the homeotic genes AG and AP3,suggesting that LEAFY plays a role in controlling the expression offloral homeotic genes.

There is increasing incentive by those working in the field of plantbiotechnology to successfully genetically engineer plants, including themajor crop varieties. One genetic modification that would beeconomically desirable would be to accelerate the flowering time of aplant. Induction of flowering is often the limiting factor for growingcrop plants. One of the most important factors controlling induction offlowering is day length, which varies seasonally as well asgeographically. There is a need to develop a method for controlling andinducing flowering in plants, regardless of the locale or theenvironmental conditions, thereby allowing production of crops, at anygiven time. Since most crop products (e.g., seeds, grains, fruits), arederived from flowers, such a method for controlling flowering would beeconomically invaluable.

SUMMARY OF THE INVENTION

The present invention arose out of the discovery that a geneticallymodified plant cell could be produced, from which a whole plant can beregenerated which stably incorporates a flower development genetic traitintroduced into the plant cell. Specifically, the trait of earlyflowering can be imparted on a plant by genetic modification accordingto the method of the invention.

In a first embodiment, the present invention provides a geneticallymodified plant comprising at least one heterologous nucleic acidsequence in its genome and characterized as having modulated floralmeristem development. Preferably, the plant is genetically modified byintroduction of a nucleic acid sequence encoding the LEAFY protein.Alternatively, the plant is genetically modified by transformation witha nucleic acid sequence encoding the LEAFY protein or a nucleic acidsequence encoding the APETALA1 protein, or both. The invention alsoprovides plant cells, plant tissue and seeds derived from thegenetically modified plant.

In a second embodiment, the invention provides a vector(s) fortransformation of a plant cell to modulate flower meristem development,wherein said vector(s) comprises a nucleic acid sequence comprising atleast one structural gene encoding a protein that modulates flowermeristem development, operably associated with a promoter. Preferably,the vector comprises a nucleic acid sequence encoding the LEAFY protein.

Also provided is a method of producing a genetically modified plantcharacterized as having modulated flower meristem development. Themethod comprises contacting a plant cell with a vector(s), comprising anucleic acid sequence comprising at least one structural gene encoding aprotein for modulating flower meristem development, operably associatedwith a promoter to obtain a transformed plant cell; producing plantsfrom said transformed plant cell; and selecting a plant exhibitingmodulated flower meristem development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration of pDW139, which is the parentalplasmid for construction of 35S::LFY vectors. Open reading frame ofLEAFY (LFY) is hatched; 5' and 3' untranslated regions are stippled.

FIG. 2 shows the early flowering phenotype of 35S::LFY tobacco plants.Left, control plant, transformed with an unrelated construct. Middle andright, two independently derived T₂ plants carrying a 35S::LFY transgene(lines 146.21, 146.26). Plants are five weeks old.

FIGS. 3A-3B show precocious enlargement of apical meristem in 35S::LFYtobacco plants. Panel (A), Control, transformed with an unrelatedconstruct. Panel (B), Experimental plant, transformed with a 35S::LFYconstruct. Size bar, 50 μm.

FIGS. 4A-4B show the early flowering phenotype of 35S::LFY Arabidopsisplants. Panel (A), Control plant, transformed with an unrelatedconstruct. The rosette leaves (rl) are significantly larger than thecotyledons (cot). Panel (B), 35S::LFY transformant (line 151.106). Thefirst two rosette leaves (rl) are smaller than the cotyledons. A smallshoot has formed, with what appear to be two cauline (=stem) leaves(cl).

FIGS. 5A-5E show the conversion of all shoots into flowers in 35S::LFYArabidopsis plants. Panel (A), For comparison, a drawing of a matureArabidopsis plant (Nossen ecotype) of about six weeks of age is shown.Panel (B), Top view of a wild-type Arabidopsis inflorescence,illustrating the indeterminacy of the shoot meristem. Panels (C)-(E)show 35S::LFY plants (generated in the Nossen ecotype), three weeks old.Panel (C), Replacement of shoots with single flowers (triangles) (line151.201). A cotyledon is indicated (cot). Panel (D), Development of aprimary terminal flower (1°) on the main shoot, and development ofsingle secondary flower (2°) in the axil of a cauline leaf (cl). Singleterminal flowers arising from the axils of curled rosette leaves (rl)are indicated by triangles (fine 151.209). Panel (E), Close-up view ofprimary and secondary flower shown in (D), at a different angle. Thegynoecium (g), comprising the carpels, appears largely normal. Thenumber of stamens (st) is reduced, and petals and sepals are absent. Asingle first-whorl organ with leaf-, sepal- and carpel-like features isindicated by an asterisk.

FIGS. 6A-6E show constitutive expression of Arabidopsis LFY convertsaspen shoots into flowers. Panels a and b show five-month-old shoots ofhybrid aspen (Populous tremula x tremuloides) grown in tissue culture.Panel a shows a 35S::LFY transformant. Solitary, lateral flowers in theaxils of leaves (If) and an abnormal terminal flower (tf) are indicated.Panel b shows a non-transgenic control. Arrowheads indicate axils ofleaves, from which lateral vegetative shoots will emerge, normally inthe following year. Note that aspen plants regenerated from tissueculture show the same juvenile phenotype during the first growing cycleas plants grown from seed (Nilsson, O., Thesis, Swedish Univ. Agricul.Sciences, 1995) Panel C is a close-up view of solitary male flower thatformed in a leaf axil of a seven-month-old 35S::LFY transformant thathad been transferred to the greenhouse. Panel d shows a close-up view ofmale flower removed from wild-type catkin shown in panel e. Note bract(b) subtending wild-type flower. Panel e shows a cluster of male catkinsof P. tremula, one of the parental species of hybrid aspen, taken from a15-year-old tree. Red pigment in anthers is apparent. Scale bars: a,b, 5mm; c, d, 1 mm; e, 20 mm.

FIGS. 7A-7D show 35S::LFY phenotype is partly suppressed by an ap1mutation. Panel a shows five-week-old plants that carry the erectamutation. The 35S::LFY AP1⁺ plant (left) has no elongated primary shoot.A primary shoot is well developed in the 35S::LFY ap1 plant (middle),although the primary shoot still terminates prematurely, and is shorterthan that of the non-transgenic ap1 plant (right). Panels b-d show adetailed view of 35S::LFY ap1 plants. Panel b shows a close-up view oflateral shoot indicated by arrowhead in panel a. Panel c shows emergingshoots in the axils of rosette leaves. Panel d shows a top view ofprimary shoot with terminal flower (tf). Panels c and d are from afour-week-old plant. The ap1 effects are enhanced further by the cal-1mutation, although there is no qualitative change in the 35S::LFY ap1phenotype.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a genetically modified plant which ischaracterized as having the phenotypic trait of early flowerdevelopment, or early flowering. The plant is genetically modified by atleast one structural gene that encodes a protein, such as LEAFY, whichis sufficient to induce flowering in the plant.

In a first embodiment, the invention provides a genetically modifiedplant comprising at least one heterologous nucleic acid sequence in itsgenome and characterized as having modulated flower meristemdevelopment. Also included herein are plant cells and plant tissue, allderived from the genetically modified plant of the invention. Inaddition, seeds which can germinate into a genetically modified plant asdescribed herein are also provided.

The term "genetic modification" as used herein refers to theintroduction of one or more heterologous nucleic acid sequences into oneor more plant cells, which can generate whole, sexually competent,viable plants. The term "genetically modified" as used herein refers toa plant which has been generated through the aforementioned process.Genetically modified plants of the invention are capable ofself-pollinating or cross-pollinating with other plants of the samespecies so that the foreign gene, carried in the germ line, can beinserted into or bred into agriculturally useful plant varieties. Theterm "plant cell" as used herein refers to protoplasts, gamete producingcells, and cells which regenerate into whole plants. Accordingly, a seedcomprising multiple plant cells capable of regenerating into a wholeplant, is included in the definition of "plant cell".

As used herein, the term "plant" refers to either a whole plant, a plantpart, a plant cell, or a group of plant cells, such as plant tissue, forexample. Plantlets are also included within the meaning of "plant".Plants included in the invention are any flowering plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants.

Examples of monocotyledonous plants include, but are not limited to,asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion,pearl millet, rye and oats. Examples of dicotyledonous plants include,but are not limited to tomato, tobacco, cotton, rapeseed, field beans,soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops orBrassica oleracea (e.g., cabbage, broccoli, cauliflower, brusselsprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash,melons, cantaloupe, sunflowers and various ornamentals. Exemplary modelsdescribed herein include the tobacco plant and the perennial tree,aspen.

The term "heterologous nucleic acid sequence" as used herein refers toat least one structural gene operably associated with a regulatorysequence such as a promoter. The nucleic acid sequence originates in aforeign species, or, in the same species if substantially modified fromits original form. For example, the term "heterologous nucleic acidsequence" includes a nucleic acid originating in the same species, wheresuch sequence is operably linked to a promoter that differs from thenatural or wild-type promoter.

As used herein, the term "nucleic acid sequence" refers to a polymer ofdeoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct. DNA encoding theproteins utilized in the method of the invention can be assembled fromcDNA fragments or from oligonucleotides which provide a synthetic genewhich is capable of being expressed in a recombinant transcriptionalunit. Polynucleotide or nucleic acid sequences of the invention includeDNA, RNA and cDNA sequences.

Examples of structural genes that may be employed in the presentinvention include the LEAFY gene and the APETALA1 gene which controlflowering. Also included in the present invention are structural andfunctional homologues of the LEAFY and APETALA1 genes. For example, inAntirrhinum majus, the snapdragon, the homologue of the LEAFY gene isthe FLORICAULA gene and the homologue of the APETALA1 gene is theSQUAMOSA gene. Other genes which control flowering will be known tothose of skill in the art or can be readily ascertained.

Nucleic acid sequences utilized in the invention can be obtained byseveral methods. For example, the DNA can be isolated usinghybridization procedures which are well known in the art. These include,but are not limited to: 1) hybridization of probes to genomic or cDNAlibraries to detect shared nucleotide sequences; 2) antibody screeningof expression libraries to detect shared structural features and 3)synthesis by the polymerase chain reaction (PCR). Sequences for specificgenes can also be found in GenBank, National Institutes of Healthcomputer database.

Hybridization procedures useful for screening for desired nucleic acidsequences utilized herein employ labeled mixed synthetic oligonucleotideprobes where each probe is potentially the complete complement of aspecific DNA sequence in the hybridization sample which includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucleic Acid Research, 9:879, 1981).

Specific DNA sequences encoding a heterologous protein of interest, suchas LEAFY protein, can also be obtained by: 1) isolation ofdouble-stranded DNA sequences from the genomic DNA; 2) chemicalsynthesis of a DNA sequence to provide the necessary codons for thepolypeptide of interest; and 3) in vitro synthesis of a double-strandedDNA sequence by reverse transcription of mRNA isolated from a eukaryoticdonor cell. In the latter case, a double-stranded DNA complement of mRNAis eventually formed which is generally referred to as cDNA.

A cDNA expression library, such as lambda gt11, can be screenedindirectly for a heterologous polypeptide having at least one epitope,using antibodies specific for the heterologous protein. Such antibodiescan be either polyclonally or monoclonally derived and used to detectexpression product indicative of the presence of heterologous proteincDNA.

A polypeptide sequence can be deduced from the genetic code, however,the degeneracy of the code must be taken into account. Nucleic acidsequences utilized in the invention include sequences which aredegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Therefore, aslong as the amino acid sequence of heterologous polypeptide results in afunctional polypeptide (at least, in the case of the sensepolynucleotide strand), all degenerate nucleotide sequences are includedin the invention.

"Modulated" flower meristem development as used herein, refers to flowerdevelopment in the plant which may be either accelerated orinhibited/delayed as compared to the naturally occurring, umnanipulatedplant. Therefore, the term "modulate" envisions the acceleration oraugmentation of flower development when development is desirable orsuppression or inhibition of flower development when development is notdesirable.

The heterologous nucleic acid sequences utilized herein are structuralgenes for flower meristem development. Preferably, such genes encode aprotein that is sufficient for the initiation of flowering, and mostpreferably, the nucleic acid sequence encodes the LEAFY protein. TheLEAFY gene or other flower meristem development gene may be utilizedalone or in combination with another structural gene, such as anothergene which encodes a protein important in the development of flowering.An example of such a gene is the APETALA1 gene.

Genetically modified plants of the present are produced by contacting aplant cell with a vector comprising a heterologous nucleic acid sequencecomprising at least one structural gene encoding a protein thatmodulates flower meristem development. To be effective once introducedinto plant cells, the structural gene of interest must be operablyassociated with a promoter which is effective in the plant cells tocause transcription of the gene of interest. Additionally, apolyadenylation sequence or transcription control sequence, alsorecognized in plant cells may also be employed. It is preferred that thevector harboring the heterologous nucleic acid sequence also contain oneor more selectable marker genes so that the transformed cells can beselected from non-transformed cells in culture, as described herein.

The term "operably associated" refers to functional linkage between apromoter sequence and the structural gene regulated by the promoternucleic acid sequence. The operably linked promoter controls theexpression of the polypeptide encoded by the structural gene.

The expression of structural genes employed in the present invention maybe driven by a number of promoters. Although the endogenous promoter ofa structural gene of interest may be utilized for transcriptionalregulation of the gene, preferably, the promoter is a foreign regulatorysequence. For plant expression vectors, suitable viral promoters includethe 35S RNA and 19S RNA promoters of CaMV (Brisson, et al., Nature,310:511, 1984; Odell, et al., Nature, 313:810, 1985); the full-lengthtranscript promoter from Figwort Mosaic Virs FMV) (Gowda, et al., J CellBiochem., 13D: 301, 1989) and the coat protein promoter to TMV(Takamatsu, et al., EMBO J 6:307, 1987). Alternatively, plant promoterssuch as the light-inducible promoter from the small subunit of ribulosebis-phosphate carboxylase (ssRUBISCO) (Coruzzi, et al., EMBO J, 3:1671,1984; Broglie, et al., Science, 224:838, 1984); mannopine synthasepromoter (Velten, et al., EMBO J, 3:2723, 1984) nopaline synthase (NOS)and octopine synthase (OCS) promoters (carried on tumor-inducingplasmids of Agrobacterium tumefaciens) or heat shock promoters, e.g.,soybean hsp17.5-E or hspl7.3-B (Gurley, et al., Mol. Cell. Biol., 6:559,1986; Severin, et al., Plant Mol. Biol., 15:827, 1990) may be used.

Promoters useful in the invention include both constitutive andinducible natural promoters as well as engineered promoters. The CaMVpromoters are examples of constitutive promoters. To be most useful, aninducible promoter should 1) provide low expression in the absence ofthe inducer; 2) provide high expression in the presence of the inducer;3) use an induction scheme that does not interfere with the normalphysiology of the plant; and 4) have no effect on the expression ofother genes. Examples of inducible promoters useful in plants includethose induced by chemical means, such as the yeast metallothioneinpromoter which is activated by copper ions (Mett, et al., Proc. Natl.Acad. Sci., U.S.A., 90:4567, 1993); In2-1 and In2-2 regulator sequenceswhich are activated by substituted benzenesulfonamides, e.g., herbicidesafeners (Hershey, et al., Plant Mol. Biol, 17:679, 1991); and the GREregulatory sequences which are induced by glucocorticoids (Schena, etal., Proc. Natl. Acad Sci., USA., 88:10421, 1991). Other promoters, bothconstitutive and inducible and enhancers will be known to those of skillin the art.

The particular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount of thestructural gene product, e.g., LEAFY, to cause early floral meristemdevelopment. The promoters used in the vector constructs of the presentinvention may be modified, if desired, to affect their controlcharacteristics.

Tissue specific promoters may also be utilized in the present invention.An example of a tissue specific promoter is the promoter expressed inshoot meristems (Atanassova, et al., Plant J., 2:291, 1992). Othertissue specific promoters useful in transgenic plants, including thecdc2a promoter and cyc07 promoter, will be known to those of skill inthe art. (See for example, Ito, et al., Plant Mol. Biol., 24:863, 1994;Martinez, et al., Proc. Natl. Acad Sci. USA, 89:7360, 1992; Medford, etal., Plant Cell, 3:359, 1991; Terada, et al., Plant Journal, 3:241,1993; Wissenbach, et al., Plant Journal, 4:411, 1993).

Optionally, a selectable marker may be associated with the heterologousnucleic acid sequence, ie., the structural gene operably linked to apromoter. As used herein, the term "marker" refers to a gene encoding atrait or a phenotype which permits the selection of, or the screeningfor, a plant or plant cell containing the marker. Preferably, the markergene is an antibiotic resistance gene whereby the appropriate antibioticcan be used to select for transformed cells from among cells that arenot transformed. Examples of suitable selectable markers includeadenosine deaminase, dihydrofolate reductase,hygromycin-B-phosphotransferase, thymidine kinase, xanthine-guaninephosphoribosyl-transferase and amino-glycoside 3'-O-phosphotransferaseII (kanamycin, neomycin and G418 resistance). Other suitable markerswill be known to those of skill in the art.

Vector(s) employed in the present invention for transformation of aplant cell to modulate flower meristem development comprise a nucleicacid sequence comprising at least one structural gene encoding a proteinthat modulates flower meristem development, operably associated with apromoter. To commence a transformation process in accordance with thepresent invention, it is first necessary to construct a suitable vectorand properly introduce it into the plant cell. The details of theconstruction of the vectors then utilized herein are known to thoseskilled in the art of plant genetic engineering.

For example, the heterologous nucleic acid sequences utilized in thepresent invention can be introduced into plant cells using Ti plasmids,root-inducing (Ri) plasmids, and plant virus vectors. (For reviews ofsuch techniques see, for example, Weissbach & Weissbach, 1988, Methodsfor Plant Molecular Biology, Academic Press, NY, Section VHI, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9, and Horsch, et al., Science, 227:1229, 1985,both incorporated herein by reference).

One of skill in the art will be able to select an appropriate vector forintroducing the heterologous nucleic acid sequence in a relativelyintact state. Thus, any vector which will produce a plant carrying theintroduced DNA sequence should be sufficient. Even a naked piece of DNAwould be expected to be able to confer the properties of this invention,though at low efficiency. The selection of the vector, or whether to usea vector, is typically guided by the method of transformation selected.

The transformation of plants in accordance with the invention may becarried out in essentially any of the various ways known to thoseskilled in the art of plant molecular biology. (See, for example,Methods of Enzymology, Vol. 153, 1987, Wu and Grossman, Eds., AcademicPress, incorporated herein by reference). As used herein, the term"transformation" means alteration of the genotype of a host plant by theintroduction of a heterologous nucleic acid sequence.

For example, a heterologous nucleic acid sequence can be introduced intoa plant cell utilizing Agrobacterium tumefaciens containing the Tiplasmid. In using an A. tumefaciens culture as a transformation vehicle,it is most advantageous to use a non-oncogenic strain of theAgrobacterium as the vector carrier so that normal non-oncogenicdifferentiation of the transformed tissues is possible. It is alsopreferred that the Agrobacterium harbor a binary Ti plasmid system. Sucha binary system comprises 1) a first Ti plasmid having a virulenceregion essential for the introduction of transfer DNA (T-DNA) intoplants, and 2) a chimeric plasmid. The latter contains at least oneborder region of the T-DNA region of a wild-type Ti plasmid flanking thenucleic acid to be transferred. Binary Ti plasmid systems have beenshown effective to transform plant cells (De Framond, Biotechnology,1:262, 1983; Hoekema, et al., Nature, 303:179, 1983). Such a binarysystem is preferred because it does not require integration into Tiplasmid in Agrobacterium.

Methods involving the use of Agrobacterium include, but are not limitedto: 1) co-cultivation of Agrobacterium with cultured isolatedprotoplasts; 2) transformation of plant cells or tissues withAgrobacterium; or 3) transformation of seeds, apices or meristems withAgrobacterium. In addition, gene transfer can be accomplished by in situtransformation by Agrobacterium, as described by Bechtold, et al., (C.R. Acad. Sci. Paris, 316:1194, 1993) and exemplified in the Examplesherein. This approach is based on the vacuum infiltration of asuspension of Agrobacterium cells.

The preferred method of introducing heterologous nucleic acid into plantcells is to infect such plant cells, an explant, a meristem or a seed,with transformed Agrobacterium tumefaciens as described above. Underappropriate conditions known in the art, the transformed plant cells aregrown to form shoots, roots, and develop further into plants. Apreferred vector(s) of the invention comprises a Ti plasmid binarysystem wherein the heterologous nucleic acid sequence encodes the LEAFYprotein. Such a vector may optionally contain a nucleic acid sequencewhich encodes a second flower development factor, such as APETALA1.Alternatively, two vectors can be utilized wherein each vector containsa heterologous nucleic acid sequence. Other flower development genes canbe utilized for construction of one or more vectors, in a similarmanner.

Alternatively, heterologous nucleic acid can be introduced into a plantcell by contacting the plant cell using mechanical or chemical means.For example, the nucleic acid can be mechanically transferred bymicroinjection directly into plant cells by use of micropipettes.Alternatively, the nucleic acid may be transferred into the plant cellby using polyethylene glycol which forms a precipitation complex withgenetic material that is taken up by the cell.

Heterologous nucleic acid can also be introduced into plant cells byelectroporation (Fromm, et al., Proc. Natl. Acad Sci., U.S.A., 82:5824,1985, which is incorporated herein by reference). In this technique,plant protoplasts are electroporated in the presence of vectors ornucleic acids containing the relevant nucleic acid sequences. Electricalimpulses of high field strength reversibly permeabilize membranesallowing the introduction of nucleic acids. Electroporated plantprotoplasts reform the cell wall, divide and form a plant callus.Selection of the transformed plant cells with the transformed gene canbe accomplished using phenotypic markers as described herein.

Another method for introducing nucleic acid into a plant cell is highvelocity ballistic penetration by small particles with the nucleic acidto be introduced contained either within the matrix of small beads orparticles, or on the surface thereof (Klein, et al., Nature 327:70,1987). Although, typically only a single introduction of a new nucleicacid sequence is required, this method particularly provides formultiple introductions.

Cauliflower mosaic virus (CaMV) may also be used as a vector forintroducing heterologous nucleic acid into plant cells (U.S. Pat. No.4,407,956). CaMV viral DNA genome is inserted into a parent bacterialplasmid creating a recombinant DNA molecule which can be propagated inbacteria. After cloning, the recombinant plasmid again may be cloned andfurther modified by introduction of the desired nucleic acid sequence.The modified viral portion of the recombinant plasmid is then excisedfrom the parent bacterial plasmid, and used to inoculate the plant cellsor plants.

In another embodiment, the invention includes a method of producing agenetically modified plant characterized as having modulated flowermeristem development, said method comprising contacting a plant cellwith a vector, comprising a heterologous nucleic acid sequencecomprising at least one structural gene encoding a protein formodulating flower meristem development, operably associated with apromoter to obtain a transformed plant cell; growing a plant from saidtransformed plant cell; and selecting a plant exhibiting modulatedflower meristem development.

As used herein, the term "contacting" refers to any means of introducingthe vector(s) into the plant cell, including chemical and physical meansas described above. Preferably, contacting refers to introducing thenucleic acid or vector into plant cells (including an explant, ameristem or a seed), via Agrobacterium tumefaciens transformed with theheterologous nucleic acid as described above.

Normally, a plant cell is regenerated to obtain a whole plant from thetransformation process. The immediate product of the transformation isreferred to as a "transgenote". The term "growing" or "regeneration" asused herein means growing a whole plant from a plant cell, a group ofplant cells, a plant part (including seeds), or a plant piece (e.g.,from a protoplast, callus, or tissue part).

Regeneration from protoplasts varies from species to species of plants,but generally a suspension of protoplasts is first made. In certainspecies, embryo formation can then be induced from the protoplastsuspension, to the stage of ripening and germination as natural embryos.The culture media will generally contain various amino acids andhormones, necessary for growth and regeneration. Examples of hormonesutilized include auxin and cytokinins. It is sometimes advantageous toadd glutamic acid and proline to the medium, especially for such speciesas corn and alfalfa. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these variablesare controlled, regeneration is reproducible.

Regeneration also occurs from plant callus, explants, organs or parts.Transformation can be performed in the context of organ or plant partregeneration. (see Methods in Enzymology, Vol. 118 and Klee, et al.,Annual Review of Plant Physiology, 38:467, 1987). Utilizing the leafdisk-transformation-regeneration method of Horsch, et al., Science,227:1229, 1985, disks are cultured on selective media, followed by shootformation in about 2-4 weeks. Shoots that develop are excised from calliand transplanted to appropriate root-inducing selective medium. Rootedplantlets are transplanted to soil as soon as possible after rootsappear. The plantlets can be repotted as required, until reachingmaturity.

In vegetatively propagated crops, the mature transgenic plants arepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenotesis made and new varieties are obtained and propagated vegetatively forcommercial use.

In seed propagated crops, the mature transgenic plants can be selfcrossed to produce a homozygous inbred plant. The inbred plant producesseed containing the newly introduced foreign gene(s). These seeds can begrown to produce plants that would produce the selected phenotype, e.g.early flowering.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells that have been transformed asdescribed. Progeny and variants, and mutants of the regenerated plantsare also included within the scope of the invention, provided that theseparts comprise the introduced nucleic acid sequences.

Plants exhibiting modulated flower meristem development can be selectedby visual observation. The invention includes a plant produced by themethod of the invention, including plant tissue, seeds, and other plantcells derived from the genetically modified plant.

In yet another embodiment, the invention provides a method formodulating flower meristem development in a plant cell, said methodcomprising contacting said plant cell with a vector as described aboveto obtain a transformed plant cell, growing the transformed plant cellunder plant forming conditions, and modulating flower meristemdevelopment in the plant. The method of the invention requires that thepromoter sequence operably linked with the structural gene. The promoteris an inducible promoter when induction of flower development isdesired. For example, a plant cell and plant is produced as describedabove and modulated flower meristem development is induced by contactingthe promoter, linked with a nucleic acid sequence encoding LEAFY, withan appropriate inducer. Such inducible promoters are described above,and include those promoters preferably inducible by chemical means.

While the present examples demonstrate that constitutive expression of afloral regulatory gene (LEAFY) causes accelerated flowering, this systemcould be modified such that flowering would be inhibited. For example,dominant-negative versions of floral regulatory genes could be expressedconstitutively. Dominant-negative mutants are proteins that activelyinterfere with the function of a normal, endogenous protein. Thus, theaction of a gene can be blocked without inactivating the structural geneitself or its RNA. This strategy has been successful for transcriptionfactors (e.g., Attardi, et al., Proc. Natl. Acad Sci. USA, 90:10563,1993; Lloyd, et al., Nature, 352:635, 1991; Logeat, et al., EMBO J.,10:1827, 1991: Mantovani, et al., J. Biol. Chem., 269:20340, 1994;Ransone, et al., Proc. Natl. Acad. Sci. USA, 87:3806, 1990; Richardson,et al., Mech. Dev., 45:173, 1994; Tsai, et al., Genes Dev., 6:2258,1992.) The LEAFY protein is likely to be a transcription factor, as itlocalizes to the nucleus and can bind to DNA in vitro. Likewise, mostother floral regulatory genes, including APETALA1, encode knowntranscription factors with a MADS DNA-binding domain (e.g., Mandel, etal., Nature, 360:273, 1992).

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLES

To study the effects of ectopic LEAFY (LFY) expression, a chimeric genein which the LFY coding region is under the control of the constitutive35S promoter from cauliflower mosaic virus (CaMV) was constructed(Odell, J. T., et al. Nature, 313:810, 1985.) By way of illustration,the chimeric 35S::LFY gene was introduced into Arabidopsis and thedistantly related tobacco plants by T-DNA mediated transformation. Otherexamples show aspen trees transformed with LFY. The phenotypic effectsobserved in transgenic plants show that LFY is not only necessary, butalso sufficient for the initiation of flower development.

EXAMPLE 1 CONSTRUCTION OF TRANSFORMATION VECTORS

For transformation of tobacco, the pDW146 vector was used. Fortransformation of Arabidopsis, the pDW151 vector was used. Both vectorsare derived from plasmid pDW139, which contains the entire open readingframe of the LEAFY (LFY) gene from Arabidopsis thaliana (Weigel, D., etal., Cell, 69:843, 1992), plus 21 bp upstream of the initiation codonand 195 bp downstream of the stop codon (for cDNA sequence, see Weigelet al., supra; genomic sequence deposited in GenBank under accessionnumber M91208). At the 5' end, a Bg12 site was added by polymerase chainreaction (Saiki, et al., Science, 239:487, 1988). At the 3' end, agenomic ScaI site was eliminated in the cloning process, and it isfollowed immediately by an Asp718 site derived from the pBluescript KS+cloning vector (FIG. 1).

FIG. 1 shows a schematic illustration of pDW139 parental plasmid forconstruction of 35S::LFY vectors. The open reading frame of LEAFY (LFY)is hatched; 5' and 3' untranslated regions are stippled.

To construct pDW146, the 1.5 kb Bg12/Asp718 fragment carrying the LFYsequences was inserted into the binary T-DNA transformation vectorpMON530 (Rogers, et al., Meth. Enzymol, 153:253, 1987), using the samesites in the vector. This vector contains an expression cassettecomprising a 0.3 kb fragment of the cauliflower mosaic virus 35Spromoter, including the transcription initiation site (Guilley, et al.,Cell, 30:763, 1982); Odell, et al, supra.); a multilinker containingseveral unique restriction sites; and a functional polyadenylationsignal from the Ti plasmid T-DNA nopaline synthase gene ("3' nos";Bevan, et al., Plant Cell, 1:141, 1983!).

To construct pDW151, the Asp7l8 site of pDW139 was filled in with Klenowenzyme (Sambrook, et al., Molecular Cloning 2nd ed. (Cold Spring Harbor:Cold Spring Harbor Laboratory, 1989) and a Bg linker was added. Theresulting Bg12 fragment was inserted into the BamHl site of pCGN18, atransformation vector containing a CaMV 35S promoter 3' nos expressioncassette (Jack, et al., Cell, 76:703, 1994).

pDW146 and pDW151 plasmid DNAs isolated from E. coli were transformedinto Agrobacterium tumefaciens strain LBA4404 (Ooms, et al., Plasmid,7:15, 1982) or ASE (Fraley, et al., Biotechnology 3:629, 1985),respectively, using the freeze-thaw method as described (Hofgen andWillmitzer, Nucl. Acids Res., 16:9877, 1988), except that LB medium(Sambrook, et al., supra) was used instead of YEB.

EXAMPLE 2 GENERATION OF TRANSGENIC TOBACCO PLANTS

For generation of transgenic tobacco plants, leaf pieces of sterilygrown tobacco strain Nicotiana tabacum var. Xanthi were infected withLBA4404/pDW146, and plants were regenerated as described (Horsch, etal., Science, 227:1229, 1985). Selection for transformed plants was with200 μg/ml kanamycin. Kanamycin resistant regenerated plants weretransferred to soil, and seeds were harvested from the primarytransformants.

FIG. 2 shows the early flowering phenotype of 35S::LFY tobacco plants.The left panel shows a control plant, transformed with an unrelatedconstruct (a LFY promoter fused to a GUS reporter gene). The middle andright panels show two independently derived T₂ plants carrying a35S::LFY transgene (lines 146.21, 146.26). The plants shown are fiveweeks old. Note abundant proliferation of leaves in the control, whilethe experimental plants have produced only two true leaves beforeinitiating a terminal flower. The insert shows a top view of floral budof plant shown at the right. The bud is still unopened.

Of 32 transgenic tobacco lines analyzed in detail, 27 exhibit the samedramatic phenotype in the progeny of the primary transformants (T₂generation). Transgenic plants develop only one pair of true leaves, inaddition to the embryonic leaves (cotyledons), before they produce aterminal flower (FIG. 2). Although wild-type tobacco plants also producea terminal flower, they generate twenty to twenty-five pairs of leavesbefore flowering. Thus, constitutive LFY expression causes precociousconversion of the shoot meristem into a floral meristem. Histologicalsections of transgenic plants reveal that the apical meristem ismorphologically different from that of untransformed plants at least asearly as five days after germination (FIG. 3). The result of thesechanges is that transformed plants produce visible floral buds after twoweeks, while normal tobacco plants flower only after about three to fivemonths (the exact time depends on environmental conditions, such aslight intensity, fertilizer, size of pots in which plants are grown,etc.).

FIG. 3 shows precocious enlargement of apical meristem in 35S::LFYtobacco plants. Panel (A) is a control, transformed with the unrelatedconstruct described in FIG. 2. Panel (B) shows an experimental plant,transformed with a 35S::LFY construct. Plants were sacrificed five daysafter germination, fixed, embedded in paraffin, and sectioned. Trianglesindicate width of meristems. Note that the leaf primordia arising at theflanks of the 35S::LFY meristem are retarded compared to those on thecontrol meristem. Size bar, 50 μm.

The precocious flowers of 35 S::LFY tobacco plants are abnormal in organidentity and organ number. The floral buds are surrounded by smallleaf-like organs, and petals are either absent or sepaloid. Stamens andcarpels are morphological normal, but their number deviates fromwild-type, being in most cases higher. Neither second-order shoots norflowers develop from the axils of the two true leaves, althoughadventitious shoots can arise from the hypocotyl.

EXAMPLE 3 GENERATION OF TRANSGENIC ARABIDOPSIS PLANTS

pDW151 was introduced into Arabidopsis by vacuum infiltration (Bechtold,et al., C.R. Acad Sci., 316:1194, 1993). Leaves of adult Arabidopsisthaliana plants of the ecotypes Wassilewskija (Ws-0) and Nossen (No-0)were infiltrated with ASE/pDW151, and seeds were harvested from theinfiltrated plants. Seeds were grown on MS medium (Murashige and Skoog,Physiol. Plant, 15:473, 1962) supplemented with 50 μg/ml kanamycin.Transformed plants were identified by their ability to grow on kanamycincontaining medium. Using this method, 27 transgenic 35S::LFY Arabidopsisplants were isolated, of which 21 exhibited essentially the samedramatic phenotype, which was very similar to that observed in 35S::LFYtobacco plants.

The transformation experiment utilized a new method that circumventstissue culture and regeneration of plants from callus, and allowsdirectly for the generation of transgenic seeds (Bechtold, et al., C.R.Acad Sci., 316:1194, 1993). In this method, leaves of adult plants arevacuum-infiltrated with a suspension of Agrobacterium cells carrying aT-DNA plasmid. The Agrobacterium cells grow in planta, where theytransfer their T-DNA to host cells, including the precursors of gameteproducing cells. Seeds were harvested from the infiltrated plants, andgrown on antibiotic containing medium to select for transformants. Asmall fraction of seeds, between one in several hundred to one inseveral thousand, were stably transformed with the T-DNA. (A singleArabidopsis plant can produce several thousand seeds.)

The following description of the 35S::LFY phenotype in Arabidopsis isbased on the analysis of first generation transformants. The phenotypeshould not change significantly in subsequent generations, because thesetransformants have been grown from seeds, as opposed to having beenregenerated from tissue culture. The same method has been used togenerate transformants with four other constructs, none of which causethe phenotype observed with the 35S::LFY construct.

FIG. 4 shows the early flowering phenotype of35S::LFY Arabidopsisplants. In panel (A), a control plant, transformed with an unrelatedconstruct. The rosette leaves (rl) are significantly larger than thecotyledons (cot). Panel (B) shows a 35 S::LFY transformant (line151.106). The first two rosette leaves (rl) are smaller than thecotyledons. A tiny shoot has formed, with what appear to be two caulineleaves (cl). The floral bud is still unopened. Both plants, which are 17days old, were selected on kanamycin containing medium for a week, whichis likely to have slowed their development somewhat.

FIG. 5 shows the conversion of all shoots into flowers in 3 5 S::LFYArabidopsis plants. Panel (A) shows a drawing of a mature Arabidopsisplant (Nossen ecotype) of about six weeks of age. Note thatindeterminate shoots develop from the axils of all rosette and stemleaves. These shoots bear a few leaves themselves, before they start toproduce flowers. Panel (B) shows a top view of a wild-type Arabidopsisinflorescence, illustrating the indeterminacy of the shoot meristem.Flowers develop in a phyllotactic spiral, with the youngest flowersbeing the closest to the center. Panels (C)-(E), 35S::LFY plants(generated in the Nossen ecotype), three weeks old. Panel (C),Replacement shoots with single flowers (triangles) (line 151.201). Acotyledon is indicated (cot). Panel (D), Development of a primaryterminal flower (1°) on the main shoot, and development of singlesecondary flower (2°) in the axil of a cauline leaf (cl). Singleterminal flowers arising from the axils of curled rosette leaves (rl)are indicated by triangles (line 151.209). Panel (E), Close-up view ofprimary and secondary flower shown in (D), at a different angle. Notethat the primary terminal flower is abnormal. The gynoecium (g),comprising the carpels, appears largely normal. The number of stamens(st) is reduced, and petals and sepals are absent. A single first-whorlorgan with leaf-, sepal- and carpel-like features is indicated by anasterisk.

35S::LFY Arabidopsis plants flower earlier than wild-type plants. Thereare only two to five rosette leaves, compared to at least eight inwild-type plants, and a stage 12 floral bud can be visible as early as17 days after germination (FIG. 4). Since it takes two weeks for thedevelopment of a stage 12 flower (Smyth, et al., Plant Cell, 2:755,1990), flowers must initiate within a few days after germination. Thisis much earlier than in wild type, where the first flowers are initiatedonly when a plant is about two weeks old. Unlike tobacco, Arabidopsishas an open inflorescence, meaning that the shoot apical meristemremains undifferentiated until the plant dies. The 35S::LFY plants notonly flower earlier, but their primary axis terminates with a singleflower, similar to the tfl mutant phenotype (see FIG. 1). Thus, ectopicexpression of LFY causes transformation of the indeterminate shootmeristem into a determinate floral meristem. In contrast with the tflmutant phenotype, no normal lateral flowers are formed before theprimary terminal flower develops (FIGS. 4B and 5D). Additional terminalflowers develop from the axils of leaves in 35S::LFY plants, indicatinga transformation of second-order shoot meristems as well (FIGS. 5C and5D). Surprisingly, most 35S: :LFY plants develop a tiny shoot, with oneor two leaves that resemble cauline (stem) leaves of wild type (FIGS. 4Band 5D). For comparison, FIG. 5A illustrates the normal architecture ofa mature Arabidopsis plant, with indeterminate shoots arising from theaxils of all leaves.

The early flowering phenotype, and the transformation of a shoot into afloral meristem, show that LFY activity is sufficient to determine theidentity of a meristem. However, since the shoot meristem producesleaves before it is converted into a floral meristem, there appear to beadditional factors that prevent the shoot meristem from responding toLFY activity immediately after germination.

35S:LFY plants appear to flower faster than any other early floweringmutant that has been described in Arabidopsis, including the embryonicflower (emf) mutant, which appears to skip the rosette phase ofvegetative development (Sung, et al., Science, 258:1645-1647, 1992).Unfortunately, the exact time of flower initiation in emf mutants hasnot been reported, but the data presented by Sung, et al., supraindicate that flower primordia are not formed before the plant is atleast nine days old, making the emf phenotype distinct from the 35S::LFYphenotype. It appears that emf mutants pause after germination, and thenproceed directly to the formation of an inflorescence.

The exact phenotype of individual 35S::LFY Arabidopsis plants varies.Most flowers observed are virtually identical to wild-type flowers(FIGS. 5D and E). Very importantly, for further analysis, stamens andcarpels are fertile. The primary terminal flowers are often abnormal, inthat the outer organs are leaf-like or absent, and the numbers of petalsis reduced, similar to the effect seen in the terminal flowers of35S::LFY tobacco plants (FIG. 5E). In addition, carpels can be unfused,and the number of stamens can be lower than the wild-type number of six.

The finding that the LEAFY gene from Arabidopsis can modify flowering intobacco implies that the mode of LEAFY function is well conserved amongflowering plants, that the Arabidopsis gene is likely to function in awide variety of flowering plants. Arabidopsis and tobacco belong to twovery divergent subclasses among the class of dicotyledonous plants.Arabidopsis is a genus within the family Brassicaceae, which belongs tothe order Capparales within the subclass Dilleniidae. Tobacco, Nicotianatabacum, belongs to the family Solanaceae, within the order Solanales ofthe subclass Asteridae. The Dilleniidae are closely related to theMagnoliidae, the most primitive subclass of dicotyledonous plants. Incontrast, the Asteridae are the most advanced subclass of dicotyledons(Cronquist, A., An Integrated System of Classification of FloweringPlants, 1981 (New York: Columbia University Press).

The two familes to which Arabidopsis and tobacco belong, Brassicaceaeand Solanaceae, are large familes of major economic importance (Heywood,V. H., Flowering Plants of the World, 1993, (New York: Oxford UniversityPress). Main crops within the Brassicaceae include oilseed rape andcabbage and its relatives, such as kale, cauliflower, broccoli, andChinese cabbage. The family Solanaceae is one of the most importantserving humankind, containing many essential vegetables and fruits suchas potatoes, tomatoes, aubergines, paprika, chilies, and bell peppers.

Recent work has shown that close homologs of Arabidopsis floralregulatory genes exist in monocotyledonous plants. For example, homologsof the APETALA1 and LEAFY genes have been identified in maize (Veit, etal., Plant Cell, 5:1205, 1993; Weigel and Meyerowitz, In Molecular Basisof Morphogenesis, pp. 91-105, 1993, (New York: Wiley-Liss).

EXAMPLE 4 CONVERSION OF ASPEN SHOOTS

Because constitutive expression of LFY can induce flowers precociouslyduring the vegetative phase of Arabidopsis, other species were examinedas well. The effect of constitutive LFY expression was studied in aperennial tree, hybrid aspen, which is derived from parental speciesthat flower naturally only after 8-20 years (Schreiner, E. J. in USDAAgriculture Handbook, 450: Seeds of Woody Plants in the United States(ed. Schopmeyer, C. S.) pp. 645-655 (U.S. Government Printing Office,Washington D.C., 1974). 35S::LFY aspen plants were obtained byAgrobacterium-mediated transformation of stem segments and subsequentregeneration of transgenic shoots in tissue culture (Nilsson, O., etal., Transgen. Res., 1:209, 1992).

Hybrid aspen was transformed as described previously (Nilsson, O., etal., ibid). Levels of LFY RNA expression were similar to those of35S::LFY Arabidopsis, as determined by Northern blot analysis. Thenumber of vegetative leaves varied between the different regeneratingshoots. Those with a higher number of vegetative leaves formed roots,allowing for transfer to the greenhouse. Individual flowers were removedeither from primary transformants that had been transferred to thegreenhouse, or from catkins collected in spring 1995 at Carlshem (Umea,Sweden) from a tree whose age was determined by counting the number ofannual rings in a core extracted with an increment borer at 1.5 m aboveground level. Flowers were fixed in formaldehyde/acetic acid/ethanol,and destained in ethanol before photography.

FIG. 6 shows that constitutive expression of Arabidopsis LFY convertsaspen shoots into flowers. Panels a and b show five-month-old shoots ofhybrid aspen (Populous tremula x tremuloides) grown in tissue culture.Panel a shows a 35S::LFY transformant. Solitary, lateral flowers in theaxils of leaves (lf) and an abnormal terminal flower (tf) are indicated.Panel b shows a non-transgenic control. Arrowheads indicate axils ofleaves, from which lateral vegetative shoots will emerge, normally inthe following year. Note that aspen plants regenerated from tissueculture show the same juvenile phenotype during the first growing cycleas plants grown from seed (Nilsson, O., supra) Panel C is a close-upview of solitary male flower that formed in a leaf axil of aseven-month-old 35S::LFY transformant that had been transferred to thegreenhouse. Panel d shows a close-up view of male flower removed fromwild-type catkin shown in panel e. Note bract (b) subtending wild-typeflower. Panel e shows a cluster of male catkins of P. tremula, one ofthe parental species of hybrid aspen, taken from a 15-year-old tree. Redpigment in anthers is apparent. Scale bars: a,b, 5 mm; c, d, 1 mm; e, 20mm.

Regenerating 35S::LFY aspen shoots initially produced solitary flowersin the axils of normal leaves (FIG. 6a,e). However, the number ofvegetative leaves is limited, and the shoot meristem is prematurelyconsumed in the formation of an aberrant terminal flower (FIG. 6a).Precocious flower development is specific to 35S::LFY transformants, assuch an effect was not observed in non-transgenic controls (FIG. 6b).Furthermore, not a single instance of precocious flower development hasbeen seen in the more than 1,500 other lines of transgenic aspen thatwere generated with various constructs during the past six years at theSwedish University of Agricultural Sciences (Nilsson O., et al., 1992supra; Nilsson, O. Thesis, Swedish University of Agricultural Sciences,1995).

Although wild-type Arabidopsis and aspen are rather different, one beinga weed and the other a tree, the overall phenotype of 35S: :LFY aspenvery much resembles that of 35S::LFY Arabidopsis. In wild-type plants ofboth species, flowers are normally formed in lateral positions oninflorescence shoots. In aspen, these inflorescence shoots are calledcatkins and arise from the leaf axils of adult trees (FIG. 6d, e). Inboth 35S::LFY Arabidopsis and 35S::LFY aspen, solitary flowers forminstead of shoots in the axils of vegetative leaves. Moreover, as inArabidopsis, the secondary shoots of trangenic aspen are more severelyaffected than the primary shoot.

An apparent LFY orthologue from poplar has been described (Strauss, S.H., et al., Mole. Breed, 1:5, 1995) which, similarly to tobacco LFY(Kelly, A., et al., PL Cell, 7:225, 1995) is already expressed duringthe vegetative stage. The vegetative expression might have suggestedthat LFY activity is not sufficient to induce flower development inthese species. The present results in aspen, which is the same in genusas poplar, indicate that this is not the case, a finding that extends totobacco, which also flowers very early when transformed with a 35S::Arabidopsis LFY construct (see Examples 1-3). One possible explanationfor the effects of 35S::LFY in these species is that the transgene isexpressed at higher effective levels in than the endogenous gene. Intobacco, expression of the endogenous gene in the center of the shootmeristem (which eventually turns into a flower meristem and forms aterminal flower) is relatively low (Kelly, A. J., et al., supra.), andit is conceivable that the other genes, such as AP1, are the primaryregulators of flower-meristem-identity in non-transgenic tobacco.

EXAMPLE 5 MEDIATION OF LEAFY ACTIVITY BY APETALA1

In addition to LFY, mutations in the genes AP1, CAL, APETALA2 (AP2) andUNUSUAL FLORAL ORGANS (UFO) are known to affect the identity ofArabidopsis flower meristems Handel, M. A., et al., Nature, 360:273,1992; Irish, V. F. & Sussex, I. M., Pl. Cell, 2:741, 1990; Jofuku, K.D., et al., Pl. Cell, 6, 1994; Levin, J. Z. & Meyerowitz, E. M. Pl.Cell, 7:529, 1995) although lfy mutations have generally the strongesteffects, and are the only ones that consistently cause a completetransformation of at least a few flowers into shoots. Mutations in allfive genes also affect the identity of floral organs, butmeristem-identity and organ-identity defects are at least in some casesseparable (Bowman, J. L., et al., Development, 119:721, 1993; Jack, T.,et al., Cell, 76:703, 1994). To determine whether any of the other genesare required to mediate the effects of 35S::LFY on meristem identity, orwhether their inactivation would merely affect the identity of floralorgans in 35S::LFY flowers, the 35S::LFY transgene was crossed intovarious mutant backgrounds.

A 35S::LFY tranformant (line DW151.117, Wassilewskija ecotype) wascrossed to ap1-1 (Landberg erecta ecotype)(Irish, F. V. & Sussex, I. M.,PI Cell 2:741, 1990.). Transheterozygote F₁ progeny was eitherbackcrossed to ap1-1 or allowed to self-fertilize. The cal genotype ofselfed F₂ progeny was determined by polymerase chain reaction(PCR)(Kempin, S. A., et al., Science, 267:552, 1994).

FIG. 7 shows that 35S::LFY phenotype is partly suppressed by an ap1mutation. Panel a shows five-week-old plants that carry the erectamutation. The 35S::LFY AP1⁺ plant (left) has no elongated primary shoot.A primary shoot is well developed in the 35S::LFY ap1 plant (middle),although the primary shoot still terminates prematurely, and is shorterthan that of the non-transgenic ap1 plant (right). Panels b-d show adetailed view of 35S::LFY ap1 plants. Panel b shows a close-up view oflateral shoot indicated by arrowhead in panel a. Panel c shows emergingshoots in the axils of rosette leaves. Panel d shows a top view ofprimary shoot with terminal flower (tf). Panels c and d are from afour-week-old plant. The ap1 effects are enhanced further by the cal-1mutation, although there is no qualitative change in the 35S: :LFY ap1phenotype.

The ap2-1, ap2-2 and ufo-2 mutations caused only additive phenotypes,and did not significantly affect the shoot-to-flower conversion in 35S:LFY plants. In contrast, the ap1-1 mutation suppressed the 35S: :LFYphenotype to a notable extent, although terminal flowers were stillformed (FIG. 7). Both the primary and secondary shoots were affected,with the strongest effects being observed in lateral positions (FIG.7b,c). The solitary flowers that develop in the axils of rosette leavesof 35S::LFY AP1⁺ plants become complex shoots with an average of 10nodes in 35S::LFY ap1 plants (FIG. 7c). These observations not onlyconfirmed that LFY can induce AP1 (which fails to become activated inearly arising flowers of lfy mutants), but also that the combinedactivities of LFY and AP1 are ever more effective in transforming shootmeristems into flower meristems than LFY activity alone.

Taken together, these results suggest that competence to respond toflower-meristem-identity genes is acquired gradually. In youngmeristems, competence appears to be low, and both LFY and AP1 arerequired to promote flower development over that of shoots. Competenceincreases later in the life cycle, and LFY alone becomes sufficient toinduce flower development.

SUMMARY

The present invention shows that constitutive expression of a singleflower meristem identity gene, such as LEY or AP1, can induce precociousflower development in plants as diverse as Arabidopsis, an ephermeralweed, and aspen, a perennial tree. The results not only contribute tothe understanding of flower development and floral induction, they arealso likely to be of interest because shorter flowering times lead toshorter generation times, which in turn allows acceleration of breedingprograms. Modem crop varieties are the result of continued improvementby breeding and two recently developed technologies have made breedingeven more important. The first technology is molecular mapping, withwhich genes encoding desirable traits can be rapidly located within thegenome. Introduction of such traits into agriculturally importantvarieties is now greatly assisted by monitoring linked molecularmarkers, instead of testing for actual expression of these traits. Thesecond technology is transformation of plants with hybrid genesconferring various traits such as engineered pathogen resistance.However, progress in this area has been delayed because the number ofplant varieties amenable to transformation is often restricted, andextensive backcrossing is needed to introgress transgenes into a desiredbackground. In both cases, marker-assisted breeding and transgeneintrogression, reduction of generation time through the induction ofprecocious flowering should prove useful.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1054                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      ctttccaattggttcataccaaagtctgagctcttctttatatctctcttgtagtttct60                 attgggggtctttgttttgtttggttcttttagagtaagaagtttcttaaaaaaggata120                aaaatgggaaggggtagggttcaattgaagaggatagagaacaagatcaatagacaagg180                acattctcgaaaagaagagctggtcttttgaagaaagctcatgagatctctgttctctt240                gatgctgaagttgctcttgttgtcttctcccataaggggaaactcttcgaatactccat300                gattcttgtatggagaagatacttgaacgctatgagaggtactcttacgccgaaagacg360                cttattgcacctgagtccgacgtcaatacaaactggtcgatggagtataacaggcttag420                gctaagattgagcttttggagagaaaccagaggcattatcttggggaagacttgcaaga480                atgagccctaaagagcttcagaatctggagcagcagcttgacactgctcttaagcacac540                cgcactagaaaaaaccaacttatgtacgagtccatcaatgagctccaaaaaaaggagag600                gccatacaggagcaaaacagcatgctttctaaacagatcaaggagagggaaaaaattct660                agggctcaacaggagcagtgggatcagcagaaccaaggccacaatatgcctccccctcg720                ccaccgcagcagcaccaaatccagcatccttacatgctctctcatcagccatctccttt780                ctcaacatgggtggtctgtatcaagaagatgatccaatggcaatgaggaatgatctcga840                ctgactcttgaacccgtttacaactgcaaccttggctgcttcgccgcatgaagcatttc900                atatatatatttgtaatcgtcaacaataaaaacagtttgccacatacatataaatagtg960                ctaggctcttttcatccaattaatatattttggcaaatgttcgatgttcttatatcaca1020               tatataaattagcaggctcctttctttttttgta1054                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 255 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyArgGlyArgValGlnLeuLysArgIleGluAsnLysIleAsn                              151015                                                                        ArgGlnValThrPheSerLysArgArgAlaGlyLeuLeuLysLysAla                              202530                                                                        HisGluIleSerValLeuCysAspAlaGluValAlaLeuValValPhe                              354045                                                                        SerHisLysGlyLysLeuPheGluTyrSerThrAspSerCysMetGlu                              505560                                                                        LysIleLeuGluArgTyrGluArgTyrSerTyrAlaGluArgGlnLeu                              65707580                                                                      IleAlaProGluSerAspValAsnThrAsnTrpSerMetGluTyrAsn                              859095                                                                        ArgLeuLysAlaLysIleGluLeuLeuGluArgAsnGlnArgHisTyr                              100105110                                                                     LeuGlyGluAspLeuGlnAlaMetSerProLysGluLeuGlnAsnLeu                              115120125                                                                     GluGlnGlnLeuAspThrAlaLeuLysHisIleArgThrArgLysAsn                              130135140                                                                     GlnLeuMetTyrGluSerIleAsnGluLeuGlnLysLysGluLysAla                              145150155160                                                                  IleGlnGluGlnAsnSerMetLeuSerLysGlnIleLysGluArgGlu                              165170175                                                                     LysIleLeuArgAlaGlnGlnGluGlnTrpAspGlnGlnAsnGlnGly                              180185190                                                                     HisAsnMetProProProLeuProProGlnGlnHisGlnIleGlnHis                              195200205                                                                     ProTyrMetLeuSerHisGlnProSerProPheLeuAsnMetGlyGly                              210215220                                                                     LeuTyrGlnGluAspAspProMetAlaMetArgAsnAspLeuGluLeu                              225230235240                                                                  ThrLeuGluProValTyrAsnCysAsnLeuGlyCysPheAlaAla                                 245250255                                                                     __________________________________________________________________________

I claim:
 1. A genetically modified plant having in its genome aheterologous nucleic acid sequence encoding a flower meristem identityprotein selected from the group consisting of LEAFY, APETALA1 and acombination thereof, wherein said plant is characterized as havingaccelerated flower meristem development.
 2. The plant of claim 1,wherein the heterologous nucleic acid sequence further comprises apromoter.
 3. The plant of claim 2, wherein the heterologous nucleic acidsequence encodes APETALA1 protein.
 4. The plant of claim 2, wherein thepromoter is a constitutive promoter.
 5. The plant of claim 2, whereinthe promoter is an inducible promoter.
 6. The plant of claim 2, whereinthe nucleic acid further comprises a selectable marker.
 7. The plant ofclaim 1, wherein the plant is a dicotyledonous plant.
 8. The plant ofclaim 1, wherein the plant is a monocotyledonous plant.
 9. A plant cellderived from the plant of claim
 1. 10. Plant tissue derived from theplant of claim
 1. 11. A seed which germinates into a plant having in itsgenome a heterologous nucleic acid sequence wherein said sequenceencodes a flower meristem identity protein selected from the groupconsisting of LEAFY, APETALA1 and a combination thereof, and whereinsaid seed is capable of germinating into a plant having acceleratedflower meristem development.
 12. A vector comprising a nucleic acidsequence comprising at least one structural gene encoding a flowermeristem identity protein selected from the group consisting of LEAFYAPETALA1, and a combination thereof, that accelerates flower meristemdevelopment, operably associated with a promoter.
 13. The vector ofclaim 12, wherein the vector comprises a T-DNA derived vector.
 14. Thevector of claim 12, wherein the structural gene encodes LEAFY protein.15. The vector of claim 14, further comprising a structural gene whichencodes APETALA 1 protein.
 16. The vector of claim 12, wherein thestructural gene encodes APETALA 1 protein.
 17. The vector of claim 12,wherein the promoter is a constitutive promoter.
 18. The vector of claim12, wherein the promoter is an inducible promoter.
 19. The vector ofclaim 18, wherein the promoter is induced by chemical means.
 20. Amethod of producing a genetically modified plant characterized as havingearly flower meristem development, said method comprising:contacting aplant cell with a vector comprising a nucleic acid sequence comprising astructural gene encoding a flower meristem identify protein selectedfrom the group consisting of LEAFY, APETALA1 and a combination thereoffor modulated flower meristem development, said gene operably associatedwith a promoter, to obtain a transformed plant cell; producing a plantfrom said transformed plant cells; and selecting a plant exhibiting saidearly flower meristem development.
 21. The method of claim 20, whereinthe contacting is by physical means.
 22. The method of claim 20, whereinthe contacting is by chemical means.
 23. The method of claim 20, whereinthe plant cell is selected from the group consisting of protoplasts,gamete producing cells, and cells which regenerate into a whole plant.24. The method of claim 20, wherein the promoter is a constitutivepromoter.
 25. The method of claim 20, wherein the promoter is aninducible promoter.
 26. A plant produced by the method of claim
 20. 27.Plant tissue derived from a plant produced by the method of claim 20.28. A method for modulating flower meristem development in a plant cellcomprising:contacting said plant cell with the vector of claim 12 toobtain a transformed plant cell; growing the transformed plant cellunder plant forming conditions; and inducing early floral meristemdevelopment in the plant under conditions and for a time sufficient toinduce the promoter of aid vector, thereby accelerating early floralmeristem development.